PROJECT SUMMARY/ABSTRACT Myotendinous junctions are the direct interface between muscle and tendon and the affected site in traumatic muscle injury and myotendinous rupture. Given the inconsistent effectiveness of existing treatments for myotendinous injuries, we propose developing an alternative approach using human induced pluripotent stem cells (iPSCs). Such cells have the capacity to create progenitor cells that can contribute to muscle and tendon regeneration. Our specific goals of this study are to engineer iPSC-derived muscle-tendon units and evaluate their utility as an in vitro model to study myotendinous junction formation, and to repair damages in muscle, tendon, and myotendinous junctions. The fundamental hypothesis guiding this proposal is that iPSC-derived musculoskeletal progenitor cells (skeletal muscle progenitor cells and tendon progenitor cells) will interact with each other and form a muscle-tendon unit with functional myotendinous junctions. This hypothesis is supported by our published studies and preliminary data demonstrating the feasibility of producing human musculoskeletal tissues from iPSCs. In this proposal, we will prepare lines of human tendon progenitor cells (hTPCs) from iPSCs. The established cells will be co-cultured with iPSC-derived skeletal muscle progenitor cells (hSMPCs) using newly featured cell culture systems for iPSCs, two-dimensional micropatterned culture platforms (Aim 1). Topographical and molecular guidance from the micropatterns can simulate cellular and molecular complexity in musculoskeletal development and pathology. Next, we will create three-dimensional muscle-tendon tissue cultures using iPSC-derived musculoskeletal progenitor cells and analyze their anatomical and physiological properties to extend the utility of iPSCs for modeling myotendinous junction formation (Aim 2). Throughout the development of these 3D culture models, we hope to identify the roles of exogenous stimulations such as signaling molecules and mechanical loads for muscle-tendon differentiation and myotendinous junction formation. Lastly, we will test the capacity of iPSC-derived muscle-tendon tissues to regenerate injured muscle, tendon, and myotendinous junctions by studying implantation in a rat model of complete myotendinous junction rupture (Aim 3). These aims will provide highly novel insights into effective approaches using iPSC-based in vitro modeling and treatments. As iPSCs can now be derived from human adult somatic tissues, this approach can be used to develop patient-specific, cell-based in vitro models and therapy for human disease. The results of this project will accelerate progress towards effective treatments for patients with musculoskeletal disorders. Given the lack of effective treatments for myotendinous injuries and the consequential burden it places on society, this study is both urgent and timely.